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The current debate about the safety of genetically modifiedfood includes some important scientific issues where morescientific data would aid the robustness of safety evaluation.One example is the possibility of gene transfer, especially fromgenetically modified plant material.

AddressesFood Safety Science Division, Institute of Food Research,Norwich Research Park, Colney, Norwich, Norfolk, NR4 7UA;e-mail: mike.gasson@bbsrc.ac.uk

Current Opinion in Biotechnology 2000, 11:505–508

0958-1669/00/$ — see front matter© 2000 Elsevier Science Ltd. All rights reserved.

AbbreviationsGI gastrointestinalGM genetically modified

IntroductionThe very public debate about the safety of geneticallymodified (GM) food has led to the voicing of safety con-cerns by a wide spectrum of individuals and groups.Although much of this lacks a credible scientific basis,some legitimate questions have been raised that warrantserious consideration. One of these is the potential fortransgenic DNA to be transferred from GM material fol-lowing its release into the environment or consumptionby man or animals. It is very well established that bac-teria have highly evolved processes for the acquisitionand rearrangement of genetic material. Arguably thebest evidence for this in nature is the development ofmultiple drug resistance, which has been analysed insome detail and represents something of a paradigm forthe roles of gene flow and DNA rearrangement in bac-terial evolution. Transfer of DNA betweenmicroorganisms in a natural environment could occur byconjugation, transformation of released genetic materialor even by bacteriophage-mediated transduction.Transfer from GM plant material is most likely to occurby transformation of released DNA.

Those charged with the regulation of GM material needto evaluate the possibility of gene transfer as well as anyconsequences. This issue became more topical with theinclusion of antibiotic-resistance genes in some GMplants intended for release into the food chain and thisfuelled debate about the existence of mechanisms bywhich DNA introduced into a transgenic plant might beacquired by bacteria. The aim of this commentary is todiscuss some recent work that aims to improve under-standing of relevant gene-transfer processes that mighttake place in nature.

Antibiotic-resistance markers Antibiotic resistance genes have been introduced into GMplants as selection markers for their primary transforma-tion. The nptII gene, which confers resistance tokanamycin and neomycin and was originally derived fromEscherichia coli transposon Tn5, is used frequently. A com-prehensive argument about the safety of nptII expressedby a plant-specific promoter was presented by Calgene [1]and accepted by the US Food and Drug Administrationand others. A significant factor is the limited importanceof both kanamycin and neomycin in the treatment of bac-terial infections in humans, mainly as a consequence oftheir relative toxicity. In addition, it is recognised thatantibiotic resistance is already widespread in bacteria andrare gene transfer from a GM food source is unlikely to beof practical consequence [2].

In contrast to nptII, several GM plants have been devel-oped in which other antibiotic-resistance genes have beenintroduced. In these cases, the antibiotic-resistance geneshave not been engineered as plant selection markers andthey retain their original bacterial promoter. Genes in thiscategory include bla, aad and nptIII. All of these genes con-fer resistance to antibiotics with greater use in clinicalmedicine than kanamycin and neomycin. In addition tothese drugs, the nptIII gene provides resistance toamikacin. The most common reason for the presence ofthese genes in GM plants is that the trait gene was firstengineered into a bacterial vector containing these antibi-otic-resistance genes using E. coli cloning. The completevector was then used to deliver the transgene by biolisticor protoplast transformation. In addition, bacterial antibiotic-resistance genes have been included in the T DNA intro-duced into transgenic plants by the Agrobacteriumtumefaciens binary vector system. These genes are notdirectly selectable in plants and there is no good reason fortheir presence in GM material destined for use as food.

Alternative selection markers that avoid the use of anti-biotic-resistance genes are becoming available andmechanisms, such as the cre/lox system [3], have beendeveloped to facilitate the removal of selection markersafter GM plant construction. Recently, Norvartis Seedsannounced their intention to phase out the use of anti-biotic-resistance genes. Their ‘Positech’ marker systemuses selection for growth on mannose and relies on a genefor phosphomannose isomerase [4]. Other approachesinclude the use of co-transformation of trait and selectiongenes followed by segregation of the latter. This has beeneffective in both Agrobacterium transformation [5] andbiolistic transformation [6]. Despite these developments,GM plant material carrying antibiotic-resistance genes

Gene transfer from genetically modified foodCommentaryMichael J Gasson

continue to be put forward for consideration by regula-tory authorities who need to assess safety on the basis ofobjective scientific criteria.

Survival of DNA One of the best-established barriers to the transfer of DNAis its sensitivity to inactivation and degradation. In the caseof food and feed, deoxyribonuclease I produced by salivaryglands, pancreas and small intestine is a potent degradativeenzyme and the low pH of the stomach or ruminant abo-masal acts to remove adenine and guanine residues,thereby eliminating biological activity [7].

Recent work on the fate of DNA in vivo has added tounderstanding of the rate at which consumed DNA isdestroyed by natural processes. Mercer et al. [8] investigat-ed the effect of human saliva on DNA survival usingcompetitive PCR and tested biological activity by measur-ing transformation into the naturally competent oralbacterium Streptococcus gordonii. Despite evidence of DNAdegradation, sufficient biologically active DNA survivedexposure to saliva to generate transformants, albeit at areduced frequency. Duggan et al. [9] investigated DNAdegradation by ovine saliva, ovine rumen fluid and silageeffluent, measuring biological activity using E. coli trans-formation. PCR amplification of DNA was possible for30 minutes after exposure to rumen fluid or silage effluent,but transforming ability was lost within one minute. Incontrast, the ability to transform E. coli was retained evenafter 24 hours exposure to saliva.

These studies suggest that DNA may remain available fortransformation in the oral cavity but be rapidly inactivatedfurther down the gastrointestinal (GI) tract. This conclu-sion has been reinforced by Chambers et al. [10] whoinvestigated the fate of the pUC18 ampicillin resistancegene in vivo using chicken feeding experiments. Bothbacteria carrying pUC18 and transgenic maize carrying thebla gene (encoding β-lactamase) were studied. PCR-restriction fragment length polymorphism (RFLP) wasused to differentiate the test bla gene from naturallyoccuring bla genes that may have been present in the GItract microflora. This was possible because the pUC18gene lacks a PstI site that is present in the natural gene.Whereas the maize-derived gene could be detected in thecrops of experimental chickens, it did not persist furtherdown the intestinal tract. In contrast, protection was pro-vided to pUC18 by the bacteria carrying it, allowing blagene detection throughout the intestinal tract.

Other work suggests that intact DNA may survive, crossthe gut epithelium, enter the blood stream and interactwith mammalian cells. Schubbert et al. [11,12] fed micewith bacteriophage M13mp18 DNA as a test molecule thatwas devoid of homology to mouse DNA. The fate of thisforeign DNA in the animals was followed by several meth-ods. Fragments of M13mp18 DNA were detected in thecontents of the small intestine, the cecum, and the large

intestine, and in the faeces and blood. It was calculatedthat 2–4% of orally administered DNA was detected in theGI tract and 0.1–0.01% was retrieved from blood.M13mp18 DNA fragments were traced by PCR to periph-eral leukocytes and located by fluorescent in situhybridisation (FISH) in about 1 of 1000 white cellsbetween 2 and 8 h after feeding and in spleen or liver cellsup to 24 h after feeding. M13mp18 DNA could be tracedby FISH in the columnar epithelial cells, in the leuko-cytes, in Peyer’s patches of the cecum wall, in liver cells,and in B cells, T cells, and macrophages from spleen.These findings suggest transport of foreign DNA throughthe intestinal wall and Peyer’s patches to peripheral bloodleukocytes and into several organs. Upon extended feed-ing, M13mp18 DNA could be cloned from total spleenDNA into a lambda vector. Schubbert et al. [13] extendedthis study and obtained similar results using a plasmidexpressing the gene for green fluorescent protein.

The significance of these results has been questioned,notably by Beever and Kemp [7]. These authors drawattention to the fact that the DNA used in these experi-ments was unmethylated and contained sequences likelyto cause up-regulation of inflammatory cell activity and tostimulate a significant immune response. It is possible thatthis induced response contributed to the detection ofDNA in white blood cells.

With respect to the survival of genetic material in the envi-ronment at large, it is well established that free DNA canbind to soil minerals [14] and plant polysaccharides [15],thereby resisting nucleolytic attack to remain available forthe transformation of competent bacteria [16].

Studies of DNA transfer from GM plant materialto bacteriaThe status of bacterial gene transfer by natural genetictransformation in the environment was extensivelyreviewed by Lorenz and Wackernagel [17]. A limited num-ber of studies have attempted to investigate DNA transferfrom GM plant material to microorganisms. These studiestend to confirm the view that such an event would beextremely rare.

Schluter et al. [18] used the plant pathogenic speciesErwinia chrysanthemi as a recipient for the transformationof plant DNA. In this model system, a transgenic potatocarrying pBR322 was used and the plant pathogenic prop-erty of Erwinia provided an intimate association betweenthe plant material and the potential bacterial recipient.The latter causes soft rot by lysing plant tissues withextracellular pectinolytic enzymes and this bacterial hostis able to support the replication and positive selection ofthe pBR322 plasmid. Evidence for plant to bacteriumtransfer was not found in this study but a series of in vitroexperiments were also undertaken and these providedquantitative data on the probability of such an event tak-ing place. This was estimated as a maximum of

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5.8 × 10–14 for an experiment using 0.9 g of potato tuberand 6.4 × 108 bacteria.

DeVries and Wackernagel [19] used naturally competentAcinetobacter and a marker rescue strategy. This studyfocused on the plant selection marker derived from thenptII kanamycin-resistance gene. The recipient bacterialstrain carried an inactive homologue of the nptII geneunder the control of a bacterial promoter. Thus, transform-ing DNA did not need to be capable of autonomousreplication or illegitimate recombination to be detected.Homologous recombination between the plant-derivednptII gene and the mutant resident gene would repair thedefect in the latter gene leading to the recovery ofkanamycin-resistant transformants. This event was detectedat a frequency of 0.9 × 10–4 per nptII gene, whereas in theabsence of recipient strain homology, transformation wasbelow the 1.3 × 10–13 limit of detection. This represents anefficient mechanism for DNA transfer in which as few as2.5 × 103 transgenic potato cells could generate a transfor-mant. Furthermore, rescue of the kanamycin-resistancemarker was effective in the presence of a more than6 × 106-fold excess of plant DNA.

It should be emphasized that this process depends onhomology and represents marker rescue rather than therecovery of unique DNA from the transgenic plant. Similarresults using marker rescue by Acinetobacter were obtainedby Gebhard and Smalla [20] using DNA from GM sugarbeet. Other studies of horizontal transfer of DNA fromplants to microorganisms have been reported by Broer et al.[21] and Hoffman et al. [22]. In addition, several as yetunpublished studies are underway that may provide directevidence for the transfer of DNA from transgenic plantmaterial to native bacteria under natural environmentalconditions. One example is DNA acquisition from GMpollen by a yeast inhabiting the intestinal tract of bees [23].The robustness of this and other preliminary findings willneed to be established, however, by peer review beforetheir significance can be judged.

The importance of selective advantageThe occurrence of a gene transfer event in itself isunlikely to be of any great significance unless it led toselective advantage in the recipient. Conversely the exis-tence of selective advantage could make even a very raregenetic event important. An example of this can be foundin lactic acid bacteria where starter bacteria have beenengineered using non-GM technology for resistance tothe bacteriophages that cause industrial problems inlarge-scale milk fermentations.

The release of starter strains carrying plasmid-encodedrestriction endonuclease and methylase genes was fol-lowed by the isolation from nature of a restriction-insensitive bacteriophage that had acquired the protectivemethylase gene by recombination in vivo [24]. Molecularanalysis of the bacteriophage genome failed to reveal an

obvious mechanism by which this recombination eventtook place and it appears to be the consequence of a rare‘illegitimate’ event. The large-scale exposure of bacterio-phage to bacteria with the restriction endonucleaseprovided a potent selection for the consequences of thisseemingly rare event to become significant. The impor-tance of selective pressure in evaluating gene transfer hasbeen emphasized by Nielsen et al. [25].

ConclusionThe possibility of gene transfer from GM constructsreleased into the environment or consumed as food is animportant aspect of safety evaluation. Clearly, gene trans-fer is very significant for bacterial evolution and itsconsequences should be the main focus for safety evalu-ation of any proposed release of GM microorganisms intofood or the environmnent. In the case of GM plants,there is controversy as to the feasibility of plant tomicroorganism transfer taking place. Recent publishedwork and the results of ongoing projects should add amore robust scientific background. Regardless of data ongene transfer, there is a case for placing more emphasis onthe consequences of any such postulated transfer eventand in this context the significance of selective advantagewarrants greater consideration.

References1. Calgene Inc.: KanR Gene: Safety and Use in the Production of

Genetically Engineered Plants. Request for an Advisory Opinion.Rockville: FDA; 1990: FDA Docket Number 90A-00416.

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3. Dale EC, Ow DW: Gene transfer with subsequent removal of theselection gene from the host genome. Proc Natl Acad Sci USA1991, 88:10558-10562.

4. Anon: Norvatis pins hopes for GM seeds on new marker system.Nature 2000, 406:924.

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7. Beever DE, Kemp CF: Safety issues associated with the DNA inanimal feed derived from genetically modified crops. A review ofscientific and regulatory procedures. Nutr Abstr Rev B – LivestockFeeds Feeding 2000, 70:175-182.

8. Mercer DK, Scott KP, Bruce-Johnson WA, Glover LA, Flint HJ: Fate offree DNA and transformation of the oral bacterium Streptococcusgordonii DL1 by plasmid DNA in human saliva. Appl EnvironMicrbiol 1999, 65:6-10.

9. Duggan PS, Chambers PA, Heritage J, Forbes JM: Survival offree DNA encoding antibiotic resistance from transgenicmaize and the transformation activity of DNA in ovine saliva,ovine rumen fluid and silage effluent. FEMS Microbiol Lett2000, in press.

10. Chambers PA, Duggan PS, Heritage J, Forbes JM: Survival of DNAfrom feedingstuffs in the gastrointestinal tract of chickens. NatBiotechnol 2000, in press.

11. Schubbert R, Lettmann C, Doerfler W: Ingested foreign (phageM13) DNA survives transiently in the gastrointestinal tract andenters the bloodstream of mice. Mol Gen Genet 1994,242:495-504.

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12. Schubbert R, Renz D, Schmitz B, Doerfler W: Foreign (M13) DNAingested by mice reaches peripheral leukocytes, spleen, and livervia the intestinal wall mucosa and can be covalently linked tomouse DNA. Proc Natl Acad Sci USA 1997, 94:961-966.

13. Schubbert R, Hohlweg U, Renz D, Doerfler W: On the fate of orallyingested foreign DNA in mice: chromosomal association andplacental transmission to the fetus. Mol Gen Genet 1998,259:569-576.

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16. Chamier B, Lorenz MG, Wackernagel W: Natural transformation ofAcinetobacter calcoaceticus by plasmid DNA absorbed on sandand groundwater aquifer material. Appl Environ Microbiol 1993,59:1662-1667.

17. Lorenz MG, Wackernagel W: Bacterial gene transfer by natural genetictransformation in the environment. Microbiol Rev 1994, 58:563-602.

18. Schluter K, Futterer J, Potrykus I: “Horizontal” gene transfer from atransgenic potato line to a bacterial pathogen (Erwiniachysanthemi) occurs — if at all — at an extremely low frequency.Biotechnology 1995, 13:1094-1098.

19. De Vries J, Wackernagel W: Detection of nptII (kanamycinresistance genes in genomes of transgenic plants by marker-rescue transformation. Mol Gen Genet 1998, 257:606-613.

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21. Broer I, Droge-Laser W, Pretorius-Guth L-M, Puhler A: Horizontalgene transfer from transgenic plants to associated soil bacteria.In Horizontal Gene Transfer – Mechanisms and ImplicationsWorkshop. Bielefeld, Germany; 1994.

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24. Hill C, Miller LA, Klaenhammer TR: In vivo genetic exchange of afunctional domain from a Type II A methylase betweenlactococcal plasmid pTR2030 and a virulent bacteriophage.J Bacteriol 1991, 173:4363-4370.

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